Contribution and Effects of PM2.5-Bound Lead to the Cardiovascular Risk of Workers in a Non-Ferrous Metal Smelting Area Considering Chemical Speciation and Bioavailability

Lead is known to have toxic effects on the cardiovascular system. Owing to its high concentration, transmission range, and absorption efficiency in organisms, inhalation of fine particulate matter (PM2.5)-bound lead (PM2.5-Pb) may cause significant cardiovascular damage. However, the contribution and adverse effects of PM2.5-Pb on workers and residents in non-ferrous metal smelting areas are not fully understood. In this work, the concentration and chemical speciation of PM2.5-Pb were analyzed to determine its pollution characteristics at a typical non-ferrous metal smelting site. A panel study conducted among factory workers revealed that PM2.5-Pb exposure makes an important contribution to the human absorption of Pb. Although the chemical speciation of PM2.5-Pb suggested poor water solubility, a high bioavailability was observed in mice (tissue average value: 50.1%, range: 31.1–71.1%) subjected to inhalation exposure for 8 weeks. Based on the bioavailability data, the relationship between PM2.5-Pb exposure and cardiovascular damage was evaluated in animal simulation experiments. Finally, a damage threshold and cardiovascular-specific risk assessment model were established for the non-ferrous metal smelting area. Our project not only accurately estimates the risk of PM2.5-bound heavy metals on the cardiovascular system but also offers a scientific basis for future prevention and therapy of PM2.5-Pb-related diseases.


S1. The specific preprocessing and pre-detection methods of filter membranes
The gravimetric analysis based on filter collection was used to confirm the mass concentration of PM 2.5 .All filter membranes were weighed using a micro-electronic balance with an accuracy of 0.01 mg before and after sampling.Ultimately, took the average of three parallel data as the final result, then used the following formula to calculate.
(1)  = ( 2 - 1 ) × 10 6  0 where stands for the mass concentration of PM 2.5 in the atmosphere (μg/m 3 ),   1 stands for membrane quality before sampling (g), stands for membrane quality  2 after sampling (g), is the sampling volume converted by standard conditions (m 3 ). 0 In order to determine the content of main anions in PM 2.5 , we further treated the filter membrane.After ultrasonic extraction in water bath at 70°C for 3 hour, eight water-soluble inorganic ions (K + , Ca 2+ , Na + , Mg 2+ , Cl -, SO 4 2-, NO 3 -, NH 4 + ) in the supernatant were determined by ion chromatography like the previous study.The contents of elemental carbon and organic carbon were quantitatively determined by total carbon analyzer 1 .S2. Quality assurance and quality control (QA/QC) ICP-MS detection followed the principle of multiple repetitions.The internal standard correction quantitative analysis method was used for determination.Each sample (including atmospheric sampling filter membrane, human urine, skin wipe, food, drinking water, mouse tissue, mouse urine and other samples) was guaranteed to have three repetitions and be detected three times.Samples digestion procedures were carried out under the same conditions of filter blank, reagent blank and high recovery rate.All samples are the same rinse method, transfer method, digestion power and digestion time to ensure that three replications of the one sample were obtained after digestion.
The digestion solution of all samples was pumped into the atomization system by a single-tube peristaltic pump and then entered the plasma torch flame.Before each detection, pure water and 2% nitric acid were used to wash the peristaltic pump for 2 min before the next sample injection.According to the operating rules of ICP-MS instrument, the instrument was adjusted to the best working state.The relevant parameters were set and the peak-hopping mode was used for data acquisition.The specific parameters were shown in Table S1.When the instrument was stable, the standard solution, blank solution and sample solution can be determined in sequence.
The recovery was 95.0% and the accuracy was 3%.The relative percentage difference of parallel samples was within 20.0 % to ensure the accuracy of experimental data.

S3. SEM of the simulated PM 2.5 suspension
Scanning electron microscope (SEM) was used to confirm the particle size of insoluble lead compounds in simulated PM 2.5 suspension.Figure S2 and Figure S3 show the morphology of the particle suspension obtained at a magnification of 1.00 kx and 2.00 kx, respectively.

S4. Calculation of exposure dose in animal experiment
We mixed standard powder according to the composition ratio of various lead compounds in PM 2.5 -Pb, 0.001g and 0.08g of the mixture were dispersed into 100mL 0.9 % normal saline to obtain the drip suspension of M-group and H 2 -group, and then diluted according to the concentration of other groups.The specific configuration concentration is shown in the Table S2.

S5. Echocardiogram of the mice of H 2 -group and C-group
Following the 8 weeks exposure period, mice were screened by echocardiography with a VisualSonics Vevo 2100 high-frequency, high-resolution micro-ultrasound system (with color-Doppler mode).Cardiac function was evaluated by noninvasive ultrasound echocardiography under light isoflurane sedation (0.5-1.00%) to prevent movement and cardio depression 2 .Mice were evaluated to obtain heart dimension and function measurements, including IVS (interventricular septum, systolic and diastolic), LVID (left ventricular internal diameter, systolic and diastolic), LVPW (left ventricular posterior wall, systolic and diastolic).EF and FS of the mice in H 2 -group and C-group were calculated by this dimension and measurements.The results are shown in the Table S3.

S6. X-ray diffraction (XRD) analysis process
Speciation of lead compounds in PM 2.5 -Pb requires XRD patterns and the powder diffraction card (PDF-2 2004) 3 .Table S5 and Table S6 show the speciation and mass proportion of lead in PM 2.5 .First of all, according to the XRD analysis, we find out the four materials which have the great match with the three major XRD diffraction peaks, and get their RIR values through the powder diffraction card.Their respective mass ratios are calculated from the RIR value and the strongest peak of each substance.
Our study and most publications have reported that the chemical composition of PM 2.5 -Pb was highly similar to that of the dust at the non-ferrous metal smelting sites.Therefore, we added other four non-ferrous metal smelting sites located in Sichuan province (Southwestern China), Hunan province (Central China), Guangdong province (Southern China) and Guizhou province (Southwestern China), respectively.By analyzing the Pb speciation in these sites, we found that the main speciation of lead in dust from these sites was consistent with that in our study (Figure . S4).
At the meantime, comparison of site PM 2.5 samples with background area samples is presented in Figure S5.

S8. The calculation process of RfD
We evaluated the cardiovascular damage by measuring heart rate, blood pressure, and urinary 8-OHdG levels in mice, where we take the last week of data to assess the final exposure effect.Through the index difference research of each group and C-group mice in the last week, the difference rate of different indicators (exceed the standard value) and exposure concentration linear fitting, through the fitting curve to find NOAELs 4 .Coincidentally, the NOAELs for the three effects are all between the concentrations in L 1/5 -group and M-group.Through the calculation of the Eq. ( 2), we obtained the reference doses corresponding to the three damage levels are 4.33×10 -3 , 4.44×10 -3 and 4.42×10 -3 mg•kg -1 •day -1 , respectively.Finally, we identified the minimum value above, 4.33×10 -3 mg•kg -1 •day -1 , as the NOAEL of PM 2.5 -Pb to human cardiovascular system in smelting sites, which slightly higher than the NOAEL (3.52×10 -3 mg•kg -1 •day -1 ) of lead (Pb) 5 . (2) where is the reference dose of PM 2.5 -Pb (mg•kg -1 •day -1 ), is   2.5 - the No Observed Adverse Effect Level (mg•kg -1 •day -1 ), is the modifying factor  (set to 1), is the uncertainty factor (assumed to be 1 because we accounted for a  difference between human and experimental animals in the exposure experiment).Figure S7.Lead content in blood-male and female.
specific preprocessing and pre-detection methods of filter membranes S2.Quality assurance and quality control (QA/QC) S3.SEM of the simulated PM 2.5 suspension S4.Calculation of exposure dose in animal experiment S5.Echocardiogram of the mice of H 2 -group and C-group S6.X-ray diffraction (XRD) analysis process S7.The chemical composition of Pb in PM 2.5 , dust and tailings heaps S8.The calculation process of RfD

Figure S1 -Figure S7 Figure S1 .
Figure S1 -Figure S7 Figure S1.The distribution of sampling points at the non-ferrous metal smelting site Figure S2.An SEM image of simulated PM 2.5 suspension at a magnification of 1.00 kx Figure S3.An SEM image of simulated PM 2.5 suspension at a magnification of 2.00 kx Figure S4.The main speciation of lead in the dust from the non-ferrous metal smelting sites located in the Gansu province (Northwestern China), Sichuan province (Southwestern China), Hunan province (Central China), Guangdong province (Southern China) and Guizhou province (Southwestern China), respectively.

Figure S5 .
Figure S5.The main speciation of PM 2.5 -Pb in the non-ferrous metal smelting site and background area Figure S6.Linear fitting of the dose of simulated PM 2.5 -Pb and lead content in tissue Figure S7.Lead content in blood-male and female

Figure
Figure

Table S3 :
The ejection fraction and fractional shortening measured in H 2 -group and Cgroup

Table S4 :
Comparison of the PM 2.5 -Pb value between study site and national city in China

Table S5 :
Approximate mass ratio of PM 2.5 -Pb compounds measured in sites by season

Table S6 :
Existence forms and mass proportion of PM 2.5 -Pb according to the XRD results

Table S7 :
Characteristics of panel study participants

Table S8 :
Concentrations of lead in various tissues of C57BL/6 mice in five exposure groups

Table S9 :
Occupational physical examination report, questionnaire analysis based on 93 workers